Tobacco heating system has less impact on bone metabolism than cigarette smoke.

Cigarette smoking promotes osteoclast activity, thus increasing the risk of secondary osteoporosis, leading to osteoporosis-associated fracture and impaired fracture healing. Heated tobacco products (HTP) are considered potential reduced-risk alternatives to cigarettes. However, their impact on bone metabolism remains to be elucidated. We developed an in vitro model that mimics in vivo bone cell interactions to comparatively evaluate the effects of HTPs and cigarette smoke on bone cell functionality and viability. We generated an in vitro coculture system with SCP-1 and THP-1 cells (1:8 ratio) cultured on a decellularized Saos-2 matrix with an optimized coculture medium. We found that, following acute or chronic exposure, particulate matter extract from the aerosol of an HTP, the Tobacco Heating System (THS), was less harmful to the bone coculture system than reference cigarette (1R6F) smoke extract. In the fracture healing model, cultures exposed to the THS extract maintained similar osteoclast activity and calcium deposits as control cultures. Conversely, smoke extract exposure promoted osteoclast activity, resulting in an osteoporotic environment, whose formation could be prevented by bisphosphonate coadministration. Thus, THS is potentially less harmful than cigarette smoke to bone cell differentiation and bone mineralization - both being crucial aspects during the reparative phase of fracture healing.

Maqui Berry and Ginseng Extracts Reduce Cigarette Smoke-Induced Cell Injury in a 3D Bone Co-Culture Model.

Cigarette smoking-induced oxidative stress has harmful effects on bone metabolism. Maqui berry extract (MBE) and ginseng extract (GE) are two naturally occurring antioxidants that have been shown to reduce oxidative stress. By using an osteoblast and osteoclast three-dimensional co-culture system, we investigated the effects of MBE and GE on bone cells exposed to cigarette smoke extract (CSE). The cell viability and function of the co-culture system were measured on day 14. Markers of bone cell differentiation and oxidative stress were evaluated at gene and protein levels on day 7. The results showed that exposure to CSE induced osteoporotic-like alterations in the co-culture system, while 1.5 µg/mL MBE and 50 µg/mL GE improved CSE-impaired osteoblast function and decreased CSE-induced osteoclast function. The molecular mechanism of MBE and GE in preventing CSE-induced bone cell damage is linked with the inhibition of the NF-κB signaling pathway and the activation of the Nrf2 signaling pathway. Therefore, MBE and GE can reduce CSE-induced detrimental effects on bone cells and, thus, prevent smoking-induced alterations in bone cell homeostasis. These two antioxidants are thus suitable supplements to support bone regeneration in smokers.

An Overlooked Bone Metabolic Disorder: Cigarette Smoking-Induced Osteoporosis.

Cigarette smoking (CS) leads to significant bone loss, which is recognized as an independent risk factor for osteoporosis. The number of smokers is continuously increasing due to the addictive nature of smoking. Therefore it is of great value to effectively prevent CS-induced osteoporosis. However, there are currently no effective interventions to specifically counteract CS-induced osteoporosis, owing to the fact that the specific mechanisms by which CS affects bone metabolism are still elusive. This review summarizes the latest research findings of important pathways between CS exposure and bone metabolism, with the aim of providing new targets and ideas for the prevention of CS-induced osteoporosis, as well as providing theoretical directions for further research in the future.

Aligned nanofiber nerve conduits inhibit alpha smooth muscle actin expression and collagen proliferation by suppressing TGF-ß1/SMAD signaling in traumatic neuromas.

Transforming growth factor-beta 1 (TGF-ß1) is a powerful activator of connective tissue synthesis that is strongly associated with the pathophysiology of traumatic neuroma. Previous studies have demonstrated that aligned nanofiber conduits made from silk fibroin and poly (L-lactic acid-co-ε-caprolactone; PLCL) could prevent traumatic neuromas. In the present study, the possible mechanisms of conduits in treating traumatic neuromas were investigated to provide theoretical basis for procedures. Aligned nanofiber conduits were used for nerve capping. Sciatic nerves of Sprague-Dawley rats were used to create an animal model. The present study contains two parts, each including four experimental groups. SB-431542/SRI-011381 hydrochloride was used to suppress/enhance TGF-ß1/SMAD signaling. Part I discussed the connections between traumatic neuroma and the proliferation of alpha smooth muscle actin (α-SMA) and collagen; it also investigated the therapeutic effect of conduits. Part II hypothesized that conduits suppressed TGF-ß1/SMAD signaling. Histological characteristics, quantitative analysis of α-SMA, collagens and signaling-related parameters were assessed and compared among groups one month postoperatively. Results from Part I demonstrated that aligned nanofiber conduits suppressed the expression of α-SMA and collagens; and results from Part II revealed the downregulation of pathway-related proteins, suggesting that the suppression was mediated by TGF-ß1/SMAD signaling. Aligned nanofiber conduits may be effective nerve capping biomaterials. One of the mechanisms involves suppressing TGF-ß1/SMAD signaling. Novel treatments using aligned nanofiber conduits could be developed to manage traumatic neuromas.

Update on Novel Non-Operative Treatment for Osteoarthritis: Current Status and Future Trends.

Osteoarthritis (OA) is a leading cause of pain and disability which results in a reduced quality of life. Due to the avascular nature of cartilage, damaged cartilage has a finite capacity for healing or regeneration. To date, conservative management, including physical measures and pharmacological therapy are still the principal choices offered for OA patients. Joint arthroplasties or total replacement surgeries are served as the ultimate therapeutic option to rehabilitate the joint function of patients who withstand severe OA. However, these approaches are mainly to relieve the symptoms of OA, instead of decelerating or reversing the progress of cartilage damage. Disease-modifying osteoarthritis drugs (DMOADs) aiming to modify key structures within the OA joints are in development. Tissue engineering is a promising strategy for repairing cartilage, in which cells, genes, and biomaterials are encompassed. Here, we review the current status of preclinical investigations and clinical translations of tissue engineering in the non-operative treatment of OA. Furthermore, this review provides our perspective on the challenges and future directions of tissue engineering in cartilage regeneration.

A simple method for decellularizing a cell-derived matrix for bone cell cultivation and differentiation.

The extracellular matrix regulates cell survival, proliferation, and differentiation. In vitro two-dimensional cell experiments are typically performed on a plastic plate or a substrate of a single extracellular matrix constituent such as collagen or calcium phosphate. As these approaches do not include extracellular matrix proteins or growth factors, they fail to mimic a complex cell microenvironment. The cell-derived matrix is an alternative platform for better representing the in vivo microenvironment in vitro. Standard decellularization of a cell-derived matrix is achieved by combining chemical and physical methods. In this study, we compared the decellularization efficacy of several methods: ammonium hydroxide, sodium dodecyl sulfate (SDS), or Triton X-100 with cold or heat treatment on a matrix of Saos-2 cells. We found that the protocols containing SDS were cytotoxic during recellularization. Heat treatment at 47 °C was not cytotoxic, removed cellular constituents, inactivated alkaline phosphatase activity, and maintained the levels of calcium deposition. Subsequently, we investigated the differentiation efficiency of a direct bone coculture system in the established decellularized Saos-2 matrix, an inorganic matrix of calcium phosphate, and a plastic plate as a control. We found that the decellularized Saos-2 cell matrix obtained by heat treatment at 47 °C enhanced osteoclast differentiation and matrix mineralization better than the inorganic matrix and the control. This simple and low-cost method allows us to create a Saos-2 decellularized matrix that can be used as an in vivo-like support for the growth and differentiation of bone cells.

Maqui berry extract prevents cigarette smoke induced oxidative stress in human osteoblasts in vitro.

Oxidative stress which can be induced by cigarette smoke (CS) is associated with an altered osteoblast differentiation, and an inhibition of the mineralization process. Therefore, treatments focusing on reducing oxidative stress in osteoblasts could be a potential therapy supporting bone formation. Maqui berry extract (MBE) is the richest natural source of delphinidins with high antioxidant activity. In the present study, we pre-/ co-/ post-incubated MBE in cigarette smoke extract (CSE)-affected human osteoblasts (hOBs), to investigate the effects of MBE as an antioxidant on hOBs. Our results clearly showed that high concentrations of MBE are toxic for hOBs, while physiological concentrations of MBE have no negative effects in vitro. Physiological concentrations of MBE can reduce oxidative stress caused by CSE in hOBs by activating the antioxidative regulator Nrf2 and its regulated antioxidative enzymes. Moreover, the physiological concentration of MBE prevents the detrimental effects of CSE-induced oxidative damage on hOBs by increasing cell viability, differentiation capability and matrix mineralization. Pre-incubation with MBE showed a positive effect on the activation of the cellular antioxidant system in hOBs. Thus, we conclude that MBE at physiological concentrations can effectively protect osteoblasts from oxidative stress-induced damage by activating the cells' antioxidative defense system.

Wnt signalling pathway in bladder cancer.

Bladder cancer (BC) is one of the most common tumours of the urinary system and is also known as a highly malignant tumour. In addition to conventional diagnosis and treatment methods, recent research has focused on studying the molecular mechanisms related to BC, in the hope that new, less toxic and effective targeted anticancer drugs and new diagnostic markers can be discovered. It is known that the Wingless (Wnt) signalling pathway and its related genes, proteins and other substances are involved in multiple biological processes of various tumours. Clarifying the contribution of the Wnt signalling pathway in bladder tumours will help establish early diagnosis indicators, develop new therapeutic drugs and evaluate the prognosis for BC. This review aims to summarise previous studies related to BC and the Wnt signalling pathway, with a focus on exploring the participating substances and their mechanisms in the regulation of the Wnt signalling pathway to better determine how to promote new chemotherapeutic drugs, potential therapeutic targets and diagnostic biomarkers.

Bisphosphonates Reduce Smoking-Induced Osteoporotic-Like Alterations by Regulating RANKL/OPG in an Osteoblast and Osteoclast Co-Culture Model.

Co-culture models have become mandatory for obtaining better insights into bone homeostasis, which relies on the balance between osteoblasts and osteoclasts. Cigarette smoking (CS) has been proven to increase the risk of osteoporosis; however, there is currently no proven treatment for osteoporosis in smokers excluding cessation. Bisphosphonates (BPs) are classical anti-osteoclastic drugs that are commonly used in examining the suitability of bone co-culture systems in vitro as well as to verify the response to osteoporotic stimuli. In the present study, we tested the effects of BPs on cigarette smoke extract (CSE)-affected cells in the co-culture of osteoblasts and osteoclasts. Our results showed that BPs were able to reduce CSE-induced osteoporotic alterations in the co-culture of osteoblasts and osteoclasts such as decreased matrix remodeling, enhanced osteoclast activation, and an up-regulated receptor activator of nuclear factor (NF)-kB-ligand (RANKL)/osteoprotegerin (OPG) ratio. In summary, BPs may be an effective alternative therapy for reversing osteoporotic alterations in smokers, and the potential mechanism is through modulation of the RANKL/OPG ratio.

Assessment of tobacco heating system 2.4 on osteogenic differentiation of mesenchymal stem cells and primary human osteoblasts compared to conventional cigarettes.

BACKGROUND: Cigarette smoking (CS) is the most common method of consuming tobacco. Deleterious effects on bone integrity, increased incidence of fractures, and delayed fracture healing are all associated with CS. Over 150 of the 6500 molecular species contained in cigarette smoke and identified as toxic compounds are inhaled by CS and, via the bloodstream, reach the skeletal system. New technologies designed to develop a reduced-risk alternative for smokers are based on electronic nicotine delivery systems, such as e-cigarettes and tobacco heating systems (THS). THS are designed to heat tobacco instead of burning it, thereby reducing the levels of harmful toxic compounds released. AIM: To examine the effects of THS on osteoprogenitor cell viability and function compared to conventional CS. METHODS: Human immortalized mesenchymal stem cells (n = 3) and primary human pre-osteoblasts isolated from cancellous bone samples from BG Unfall Klinik Tübingen (n = 5) were osteogenically differentiated in vitro with aqueous extracts generated from either the THS 2.4 "IQOS" or conventional "Marlboro" cigarettes for up to 21 d. Cell viability was analyzed using resazurin conversion assay (mitochondrial activity) and calcein-AM staining (esterase activity). Osteogenic differentiation and bone cell function were evaluated using alkaline phosphatase (AP) activity, while matrix formation was analyzed through alizarin red staining. Primary cilia structure was examined by acetylated α-tubulin immunofluorescent staining. Free radical production was evaluated with 2',7'-dichlorofluorescein-diacetate assay. RESULTS: Our data clearly show that THS is significantly less toxic to bone cells than CS when analyzed by mitochondrial and esterase activity (P < 0.001). No significant differences in cytotoxicity between the diverse flavors of THS were observed. Harmful effects from THS on bone cell function were observed only at very high, non-physiological concentrations. In contrast, extracts from conventional cigarettes significantly reduced the AP activity (by two-fold) and matrix mineralization (four-fold) at low concentrations. Additionally, morphologic analysis of primary cilia revealed no significant changes in the length of the organelle involved in osteogenesis of osteoprogenitor cells, nor in the number of ciliated cells following THS treatment. Assessment of free radical production demonstrated that THS induced significantly less oxidative stress than conventional CS in osteoprogenitor cells. CONCLUSION: THS was significantly less harmful to osteoprogenitor cells during osteogenesis than conventional CS. Additional studies are required to confirm whether THS is a better alternative for smokers to improve delays in bone healing following fracture.

Material-Dependent Formation and Degradation of Bone Matrix-Comparison of Two Cryogels.

Cryogels represent ideal carriers for bone tissue engineering. We recently described the osteogenic potential of cryogels with different protein additives, e.g., platelet-rich plasma (PRP). However, these scaffolds raised concerns as different toxic substances are required for their preparation. Therefore, we developed another gelatin (GEL)-based cryogel. This study aimed to compare the two scaffolds regarding their physical characteristics and their influence on osteogenic and osteoclastic cells. Compared to the PRP scaffolds, GEL scaffolds had both larger pores and thicker walls, resulting in a lower connective density. PRP scaffolds, with crystalized calcium phosphates on the surface, were significantly stiffer but less mineralized than GEL scaffolds with hydroxyapatite incorporated within the matrix. The GEL scaffolds favored adherence and proliferation of the osteogenic SCP-1 and SaOS-2 cells. Macrophage colony-stimulating factor (M-CSF) and osteoprotegerin (OPG) levels seemed to be induced by GEL scaffolds. Levels of other osteoblast and osteoclast markers were comparable between the two scaffolds. After 14 days, mineral content and stiffness of the cryogels were increased by SCP-1 and SaOS-2 cells, especially of PRP scaffolds. THP-1 cell-derived osteoclastic cells only reduced mineral content and stiffness of PRP cryogels. In summary, both scaffolds present powerful advantages; however, the possibility to altered mineral content and stiffness may be decisive when it comes to using PRP or GEL scaffolds for bone tissue engineering.

An aligned nanofiber nerve conduit that inhibits painful traumatic neuroma formation through regulation of the RhoA/ROCK signaling pathway.

OBJECTIVE: Traumatic neuromas represent a prevalent source of neuropathic pain. As of yet, there has been no single treatment method that can guarantee permanent relief of symptoms. Although nerve-capping techniques have shown promise, their exact mechanisms remain elusive. The authors' aim was to examine the role of the RhoA/ROCK signaling pathway in the prevention of neuroma formation after neurectomy utilizing a nerve-capping technique. METHODS: An aligned nanofiber tube was fabricated to cap the sciatic nerve in Sprague Dawley rats. The rats (n = 60) were randomly divided into the aligned SF/P (LLA-CL) capping group (capping group, n = 20), the capping and Y-27632 (ROCK pathway inhibitor) intervention group (intervention group, n = 20), and the no-capping group (control group, n = 20). The authors undertook a comprehensive assessment of the capping group, examining the animals' behavior, the extent of neuroma development, histology, gene and protein expression, and ultrastructural changes associated with the RhoA/ROCK signaling pathway. These findings were compared with those in the intervention and control groups. RESULTS: The inciting injury resulted in the expression of the RhoA/ROCK signaling pathway, as well as its further upregulation in peripheral neurons. Axon outgrowth was significantly increased when RhoA/ROCK signaling pathway was suppressed. The average autotomy score in the capping group was observed to be much lower than that of the intervention and control groups. At 30 days postneurectomy, the capping group displayed no obvious neuroma formation, while a bulbous neuroma was found in the nerve stumps of both the control and intervention groups. Quantitative real-time polymerase chain reaction and the Western blot analysis demonstrated that the expression of myelin-associated glycoprotein was substantially upregulated in the capping group; in contrast, the expression of NF-200 was significantly downregulated. The expression of myosin light chain was notably lower in the intervention group, but there was no significant difference when compared with the control group (p > 0.05). CONCLUSIONS: The RhoA/ROCK signaling pathway has emerged as a critical player in the process of traumatic neuroma formation after neurectomy. It is possible that the nerve-capping technique could generate a "regenerative brake" based on the regulation of the RhoA/ROCK signaling pathway in this event. These findings may provide concrete evidence that could help develop new strategies for the management of painful neuromas.

Individual Treatment of Delayed Distal Biceps Tendon Rupture: Case Report and Literature Review.

INTRODUCTION: Complicated elbow injuries (elbow injuries with bone and soft tissue injury) with distal biceps tendon ruptures (DBTRs) are not uncommon. There are several treatment modalities in different situations of injuries. In this article, we reported 3 successful individual treatments of delayed DBTR with complicated elbow injuries. MATERIALS AND METHODS: Three cases of complicated elbow injuries treated between 2010 and 2016 were reviewed. The delayed DBTR cases were summarized and treated. Mayo Elbow Performance Score value, range of motion, and visual analog scale score were used to assess outcomes after a minimum follow-up of 12 months. RESULTS: All 3 patients were male, aged 47 to 54 years (mean, 49.6 years). Patients received surgical treatments. After a mean follow-up of 13.7 months, in cases 1 and 2, Mayo Elbow Performance Score values improved by 50% and 100%, elbow flexion-extension arc were 115 degrees and 110 degrees, pronation-supination arc were 130 degrees and 120 degrees. Arthrodesis case reported pain relief; visual analog scale score for pain was 0 to 1. No postoperative complications were observed, and all patients were satisfied with the results. CONCLUSIONS: Individual treatment is advised in DBTR with complicated elbow injuries. Secondary treatment of DBTR can achieve satisfactory results using individual strategies depending on patients' overall condition.

Metformin relieves neuropathic pain after spinal nerve ligation via autophagy flux stimulation.

Neuropathic pain is a well-known type of chronic pain caused by damage to the nervous system. Autophagy is involved in the development and/or progression of many diseases, including neuropathic pain. Emerging evidence suggests that metformin relieves neuropathic pain in several neuropathic pain models; however, metformin's cellular and molecular mechanism for pain relief remains unknown. In this study, we investigated the therapeutic effects of metformin on pain relief after spinal nerve ligation (SNL) and its underlying mechanism of autophagy regulation. Behavioural analysis, histological assessment, expression of c-Fos and molecular biological changes, as well as ultrastructural features, were investigated. Our findings showed that the number of autophagosomes and expression of autophagy markers, such as LC3 and beclin1, were increased, while the autophagy substrate protein p62, as well as the ubiquitinated proteins, were accumulated in the ipsilateral spinal cord. However, metformin enhanced the expression of autophagy markers, while it abrogated the abundance of p62 and ubiquitinated proteins. Blockage of autophagy flux by chloroquine partially abolished the apoptosis inhibition and analgesic effects of metformin on SNL. Taken together, these results illustrated that metformin relieved neuropathic pain through autophagy flux stimulation and provided a new direction for metformin drug development to treat neuropathic pain.

The complicated role of venous drainage on the survival of arterialized venous flaps.

The arterialized venous flap (AVF) has been gradually popularized in clinical settings; however, its survival is still inconsistent and the role of venous drainage remains elusive. In this study, we aimed to investigate the role of venous drainage on the flap survival of arterialized venous flaps. An arterialized venous flap was outlined symmetrically in the rabbit abdomen. The arterial perfusion flap with a unilateral vascular pedicle was taken as the control group and three other experimental groups (I, II and III) were designed based on the number of drainage veins (n = 1, 2 and 3 in the three groups, respectively). Compared with the control group, significant venous congestion was noted in all the experimental groups and the most severe one was seen in group I; while no statistical difference was observed between groups II and III. Similar results regarding blood perfusion state, epidermal metabolite levels and flap survival status were obtained among the three groups. These findings suggested that venous drainage is vital in the survival of the flap, but unlike in the arterial perfusion flaps, the problem of venous congestion can only be partially solved by increasing the number of draining veins. Further studies are warranted to gain insight into this complicated issue.

The Value of Postconditioning in Plastic and Reconstructive Surgery: A Systematic Review.

Background Ischemia-reperfusion (I/R) injury by abrupt restoration of circulation after prolonged ischemia has still been an unsolved problem in plastic and reconstructive surgery. The concept of postconditioning (post-con), which has been well described in cardiovascular surgery, has been recently introduced in plastic and reconstructive surgery. As an "after-injury strategy," post-con may be a promising approach to reduce I/R injury and improve flap survival after ischemia. Methods A systematic review was performed by searching electronic databases of PubMed and web of science to identify all the studies regarding the application of the post-con technique in plastic and reconstructive surgery between 1950 and 2015. Inclusion criteria were English articles with clear reporting the post-con techniques and detailed outcomes. Results In total, 476 articles were identified and 18 studies reporting post-con in plastic and reconstructive surgery met the inclusion criteria in this review, including 11 studies of mechanical post-con, 3 studies of pharmacological post-con, 1 study of both mechanical and pharmacological post-con, and 3 studies of remote post-con. All these studies reported protective effects of any kind of post-con techniques in I/R injuries and could improve flap survivals. Conclusion In general, the strategy of post-con may effectively reduce I/R injury and improve the survival of flaps after ischemia in animal studies, yet no consensus regarding the exact technical details (intervention timing, cycles, intermittent duration, etc.) has been reached. Further studies aiming to explore its mechanisms as well as specific methodology are required before clinical application in plastic and reconstructive surgery.

Significance of alpha smooth muscle actin expression in traumatic painful neuromas: a pilot study in rats.

Treatment of painful neuromas remains a challenge and the mechanism of neuroma-associated pain is not yet fully understood. In this study, we aimed to observe the expression of alpha smooth muscle actin (α-SMA) in traumatic neuromas and to investigate its possible roles in the cause of neuropathic pain in a rat model. The rat sciatic nerve was used and the experiment was divided into two parts. In part I, our results showed significantly higher levels of α-SMA and the pain marker c-fos in the autotomy group than in the no-autotomy group. In part II, the expression of α-SMA in neuromas was down- and up-regulated using SB-431542 and GW9662, respectively. A significant correlation between autotomy scores and the expression level of α-SMA was found (R = 0.957; p < 0.001) and the expression level of α-SMA was positively related to the autotomy scores (R(2) = 0.915, p < 0.001). We concluded that the expression of α-SMA plays certain roles in the neuroma-associated pain, either as a direct cause of pain or as an indirect marker of existence of local mechanical stimuli. Our findings may provide new insights into the development of new treatment modalities for the management of intractable painful neuromas.